Pain is an uncomfortable sensation caused by inflammation, nerve injury, or overly-sensitive tissue reacting to mechanical, thermal or chemical stimuli. It is a major health problem: every year many work days are lost due to pain-related conditions. Among a wide variety of pains, neuropathic pain is a disease arising from nerve damage and affects >1 million Americans. This condition arises from a variety of causes including diabetes, herpes zoster infection (chicken pox/shingles), traumatic nerve injury, cancer, or treatment of cancer with chemotherapeutic agents. Inflammatory pain is another kind of pain that constitutes the single largest category due to its multiple etiologies. The search for new analgesic therapy is an area of great interest to the medical community (Reichling & Levine, 1999) because it is associated with other local or systemic diseases, such as auto-immune disorder like rheumatoid arthritis and because it is chronic and thus causes prolonged suffering.
Most of the current pain treatment use remedies of systemically-administered drugs, such as non-steroidal anti-inflammatory drugs (NSAIDS) or opioids. Many of these drugs cause systemic sides effects ranging from the increase in heart risk to addiction. There are only a few pain remedies that use local routes of administration, such as capsaicin cream, which does not work on all kinds of pain and causes local irritation (burning sensation, skin pain, skin inflammation, etc).
Protein kinase C (“PKC”) is a key enzyme in signal transduction involved in a variety of cellular functions, including cell growth, regulation of gene expression, and ion channel activity. The PKC family of isozymes includes at least 11 different protein kinases that can be divided into at least three subfamilies based on their homology and sensitivity to activators. The families are the classical, the novel, and the atypical subfamilies. Each isozyme includes a number of homologous (“conserved” or “C”) domains interspersed with isozyme-unique (“variable” or “V”) domains. Epsilon PKC is a member of the “novel” subfamily, along with δ, η and θPKC. Members of this subfamily typically lack the C2 homologous domain and do not require calcium for activation. Individual isozymes of PKC have been implicated in the mechanisms of various disease states. Epsilon PKC inhibitory peptides derived from εPKC have been generated and shown to impact nociception. For example, see U.S. Pat. Nos. 6,376,467 and 6,686,334.
One problem with this approach is that the “naked” termini of the excised fragments are different from their context in the protein, revealing free amine and carboxyl groups at the points where the fragment attaches to the remainder of the protein. These extraneous moieties may render the peptide more susceptible to proteases. As a result of these liabilities the potency of the peptide may be less than desired and the in vivo half-life may be significantly shortened.
A second area of the prior art makes use of a similar strategy, wherein “carrier” peptides are designed as fragments of HIV-Tat and other proteins. These peptide fragments mimic the ability of the parent protein to cross cell membranes. Of particular interest is the property that “cargo” peptides can be attached to these carrier peptides such that both cargo and carrier peptides are carried into the cell by these carrier peptide fragments.
Recognizing that the carrier peptides are fragments, similar deficiencies may apply as noted above for the cargo peptides. That is, the exposed termini may confer undesirable properties including protease susceptibility.
Prior art cargo/carrier peptide constructs have made use of a Cys-Cys disulfide bond between cargo and carrier, which can be cleaved by a number of agents, such as glutathione reduction when the peptides enter cells. This property has been thought to be important for biological activity, since the physical separation of cargo and carrier allows the two moieties to exert their independent effects within the cell. However, this hypothesis has not been convincingly tested, and non-cleavable analogs may in fact have good activity. Further, the disulfide bond is cumbersome to assemble, and prone to chemical degradation.
The design of certain prior art cargo/carrier peptides is based on a contiguous sequence of amino acids from the protein. However, the optimal length of the peptide has not yet been well defined, being based on sequence comparison analysis and theoretical prediction of the desired sequence rather than on an empirical basis of analog testing. Thus, increased potency may be anticipated from analogs of the previously described cargo peptides which contain additional residues corresponding to the εPKC domain from which the have been derived.